In the field of laser-scanning microscopy (confocal microscopy, two-photon microscopy, CARS microscopy, Raman microscopy), the speed and image quality with which data can be recorded depends on the luminous efficacy in the focus of the laser. In particular with dynamic measurements, problems occur due to local photo damage. The image quality depends on the scanning process and the quality of the focus. To achieve optimal resolution, firstly, the sampling (scanning) must be less than half as large as the focus size, secondly, the number of photons per scanning dot contributing to the signal must be large enough so that on the basis of Poisson statistics, sufficiently accurate localization of the observed objects is possible. In practice, a compromise between gentle imaging, measurement speed and resolution must generally be found. With dynamic measurements, in which many images are to be recorded in sequence, it is therefore frequently necessary to select the scanning coarser than the optical resolution.
It is generally not easy to influence the size of the focus itself, because it is given inter alia by the numerical aperture of the illuminating lens. When reducing the numerical aperture by reducing illumination of the lens, the z-resolution along the beam of the microscope in many cases deteriorates significantly. It therefore happens that in many cases images with coarse pixel resolution are scanned with very fine foci, so that the focus volumes between the individual lines do not overlap and large areas of the object can not contribute to image. This underscanning, in addition to the reduced resolution, also results in the fact that a large part of the sample within the image field is not swept by the laser beam and therefore does not contribute to image intensity.
It is not possible to randomly increase the intensity of illumination, and to increase the luminous efficacy from the focus volume at random, since in many cases, in particular with nonlinear microscopy techniques requiring short laser pulses, damage depends highly non-linearly on the irradiated average output of the laser.
The only option to increase the total luminous efficacy is in parallel illumination of different volumes in the object. There is currently no possibility to dynamically adjust the volume scanned to the pixel size of the final image at any time without losing the Z-resolution.